Dihydrocinnamyl phenols useful as antimicrobial agents

ABSTRACT

SUBSTANCES WHICH ARE SUBJECT TO MICROBIAL SPOILAGE ARE PRESERVED BY ADDITION OF A DIHYDROCINNAMYL PHENOL E.G., DIHYDRO-2-CINNAMYL-PHENOL, DIHYDRO-4-CINNAMYL-PHENOL, DIHYDRO-2-CINNAMYL-4-METHYLPHENOL, ETC.

United States Patent 3,775,541 DROCINNAMYL PHENOLS USEFUL AS A i ii :I' CROBIAL AGENTS Leonard Jurd, Berkeley, Kenneth L. Stevens, Walnut Creek, and Alfred Douglas King, J12, Martinez, Calif., assignors to the United States of America as represented by the Secretary of Agriculture N0 Drawing. Filed May 25, 1972, Ser. No. 257,031 Int. Cl. A0111 9/26 US. $1. 424-346 9 Claims ABSTRACT OF THE DISCLOSURE Substances which are subject to microbial spoilage are preserved by addition of a dihydrocinnamyl phenol, 55., dihydro 2 cinnamyl-phenol, dihydro 4-cinnamyl-phenol, dihydro-2-cinnarnyl-4-rnethylphenol, etc.

Anon-exclusive, irrevocable, royalty-free license in the invention herein described, throughout the world for all purposes of the United States Government, with the power to grant sublicenses for such purposes, is hereby granted to the Government of the United States of America.

DESCRIPTION OF THE INVENTION This invention relates to the preservation of substances which are normally subject to microbial spoilage. The objects of the invention include the provision of novel processes and compositions for accomplishing such preservation. Further objects of the invention will be evident from the following description wherein parts and percentages are by weight unless otherwise specified. The abbreviation p.p.m. used herein refers to parts per million on a weight/volume (w./v.) basis. Temperatures are given in degrees centigrade. The symbol is used to designate the phenyl radical.

In accordance with the invention, it has been found that certain agents exhibit unexpected antimicrobial activity and are useful for preserving all kinds of materials which are normally subject to microbial spoilage. The agents in question are certain dihydrocinnamyl phenols, and their antimicrobial activity is unusual and unexpected because it is not shared by closely-related phenol derivatives.

Generically, the agents in accordance with the invention have the structurewhere at least one but not more than 3 of the Rs are hydroxy, and the remainder of the Rs are each independently selected from the group consisting of lower alkyl, lower alkoxy, and hydrogen.

The compounds of the invention are especially useful because they are active against many microorganisms, including those in the categories of bacteria, yeasts, and molds. In other words, the compounds are not just active against one or a few organisms; rather, they display broadspectrum antimicrobial activity.

Patented Nov. 27, 1973 Examples of particular compounds within the scope of the invention are given below by way of illustration and D1hydro-4-cinnamyl-phen0l Dihydro-2-einnamyl-phenol Dihydr0-2-clnnamyl-4-methylphenol (|)H DM Dihyclr0-4-cinnarnyl-2-methylphenol Dihydro-2-cinnamyl-4-ethylphenol....;-.;; OH

@mEh-q Dihydro-2-cinnamyl-4-n-propylphen01 (3H Hr-CHr-CH,

Dihydro-2-cinnamyl-Hsopropylphenol.. (|)H @qcmnas)| Dihydr0-4-cinnamyl-3-methyl-6- 0H isopropylphenol. I

(CHQaCH- :)a Dihydro-24:1nnamyl-t-methoxyphenol.... (IDH OCH:

Dihydro-4-elnnamyl-2 rnethoxyphenol 9H OCH;

( un-(b DlhydrM-cinnamyl-resorclnol (I)H M-e Dlhydro-2-clnnamyl-5-methoxy-quinol..-. CIJH CHZO The invention encompases not only the use of any of the above-described agents individually, but also mixtures thereof.

In preserving substances in accordance with the invention, any of the aforesaid agents or mixtures thereof are incorporated with the substance, using an amount of the agent to inhibit microbial growth. Additional conventional treatments such as dehydration, canning, refrigeration, or freezing may be applied to the substance containing the added agent. The incorporation of the agent with the substance may involve a mixing of the substance and the agent-this is especially suitable where the substance is in liquid or particulate form. Where the substance is in the form of pieces of large dimensions the agent may be incorporated therewith by coating it on the surface of the pieces. For such purpose the agent is preferably dispersed in a carrier-a liquid such as water, alcohol, wateralcohol blends, oils, or a finely-divided solid such as salt, starch, talc, or the like.

The invention is of wide versatility and can be applied for the preservation of all kinds of substances which are normally subject to microbial spoilage. Typical examples of such substances are listed below by way of example. Foodstuifs such as fruits, vegetables, juices, milk eggs, meat, fish, grains, cereal products, cheese, etc. Animal glues and mucilages; dextrins; starch pastes and solutions; cosmetic, medicinal, and dental preparations; vitamin preparations; pastes, solutions, or other preparations of natural gums such as tragacanth, Arabic, acacia, karaya, locust bean, agar-agr, pectin, algin, etc.; fermentation broths, mashes, and residues from fermentation processes; whey; wines and Vinegars; animal feeds and ingredients of animal feeds such as fish meals, blood meals, feather meal, meat scraps, bone meal, tankage, grains, and oilseed meals; proteins and protein hydrolysates; textile printing pastes; paints containing proteins or other spoilable dispersing agents; solutions of bark extracts or other tanning agents; molasses; byproducts or wastes that contain potentially valuable carbohydrate, proteinous or fat ingredients such as stick liquor, corn steep liquor, fruit cannery wastes, citrus peels, cull fruit and vegetables, tops of root vegetables, distillers slops, pulp liquors, wash water from textile de-sizing operations, waste liquors from wool scouring plants, dairy and slaughter house wastes and liquors, etc.

The compounds of the invention display activities which are equal or even superior to those of widely-used antimicrobial agents. This is illustrated by the following: In general, the compounds of the invention are superior to such agents as phenol, resorcinol, o-phenyl-phenol, and the alkali metal sorbates and benzoates. For instance, the compounds of the invention, at concentrations of 12 to 25 p.p.m., were found to inhibit the growth of four bacteria (Bacillus cereus, Sarcina lutea, Staphylococcus aurcur, and Streptococcus lactis), whereas to achieve the same effect it required to 200 p.p.m. of o-phenyl-phenol, more than 800 p.p.m. of potassium sorbate, and more than 1000 p.p.m. of sodium benzoate.

Alkyl 4-hydroxybenzoates are Well known to exhibit potent microbial activity. In general, the compounds of the invention display a superior activity as compared to these benzoates wherein the alkyl group contains less than seven carbon atoms. For instance, whereas the compounds of the invention at a concentration of 12 to 25 p.p.m. will inhibit the growth of the four bacteria noted above, it requires concentrations of 100 to 400 ppm. for the same result to be achieved with the C -C alkyl 4-hydroxybenzoates. Moreover, it may be noted that the compounds of the invention have an advantage in that their water-solubility is higher than that of the higher alkyl (e.g., heptyl) 4- hydroxybenzoates, and that the compounds of the invention do not exhibit the strong and unpleasant odor which characterizes the said benzoates. Also, with respect to some microorganisms, the compounds of the invention inhibit growth when applied at levels less than required with the higher alkyl 4-hydroxybenzoates. This is the case, for example, with bacteria such as Acaligenes fuecelis and E. coli, yeast such as Pichia chodati, Hansenula anomala, and Saccharomyces cerevisiae, and molds including Aspergillus flavus, A. niger, Penicillium chrysogenum, Rhizopus senti, Botrytis cinerae, Byssochlamys fulva, and Alternar1a sp.

Of the various compounds include within the scope of the invention, the dihydrocinnamyl-substituted monophenols display particularly high antibiotic activity against a large variety of different microorganisms, and therefore are preferred with respect to the dihydrocinnamyl derivatives of dior tri-phenols.

As evident from the explanation immediately (following Formula I above, the invention includes compounds wherein there may be nuclear lower alkyl or lower alkoxy substituents in addition to the hydroxy groups. In general, the compounds are preferred wherein such additional substituents are absent or, if present, are in small number, e.g., a total of one to two lower alkyl and/or lower alkoxy substituents. Taking the foregoing facts into con sideration, we prefer to employ the compounds of the sub-generic category represented by the formulawherein- R is lower alkyl,

R" is lower alkoxy,

n is an integer from O to 2,

m is an integer from 0 to 2, and

the sum of n and m is not more than 2.

The compounds of the invention may be synthesized by known procedures. A typical plan is to first prepare a cinnamyl-substituted phenol, and then to hydrogenate this intermediate to form the dihydrocinnamyl-substituted phenol.

The intermediate may be prepared by the procedures of Hurd et al., Jour. Am. Chem. Soc., vol. 59, pp. 107- 109; Barnes et al., Tetrahedron, vol. 21 pp. 2707-2715; Jurd, Experientia, vol. 24, pp. 858-860; Jurd, Tetrahedron, vol. 25, pp. 1407-4416; and Jurd, Tetrahedron Letters No. 33, pp. 2863-2866. Typically, these syntheses involve condensing a selected phenol (phenol itself, a cresol, resorcinol, guaiacol, hydroquinone mono- 2111612113 1 ether, etc.) with cinnamyl bromide or cinnamyl cohol.

In a next step, the cinnamyl-phenol intermediate is subjected to standard procedures to hydrogenate the ethylenic linkage whereby to produce a dihydrocinnamyl-phenol. Generally, this involves treating the cinnamylphenol with hydrogen gas in the presence of a suitable catalyst, such as platinum oxide, palladium on charcoal, and the like. Typically, l0-20 grams of a cinnamylphenol is dissolved in 50-100 milliliters of a non-hydrogenatable organic solvent, such as acetic acid, tetrahydrofuran, and the like, and 0.1 to 1.0 gram of the catalyst is added. Then, hydrogen gas is applied at approximately 30-50 pounds per square inch. The dihydro derivative can be purified by common techniques, for example, distillation or crystallization.

In the copending application of Leonard Jurd et al., Ser. No. 74,485, filed Sept. 22, 1970, now US. Pat. 3,745,- 222 issued July 10, 1973, there are described certain cinnamyl phenols which are useful as antimicrobial agents. The said cinnamyl phenols have the structure wherein at least one but not more than three of the Rs are hydroxy radicals, and the remainder of the Rs are each independently selected from the group consisting of lower alkyl, lower alkoxy, and hydrogen. It should be noted that the compounds of Ser. No. 74,485 contain the unsaturated side-chain CH CH=CH-, whereas in the compounds of the present invention the corresponding substituent is the saturated group CH CH CH As such, the instant agents represent a class of compounds totally separate and distinct from those of Ser. No. 74,485. Moreover, the compounds of the present invention provide an advantage in that they are more stable. Since they do not contain ethylenic unsaturation, they are not subject to oxidization when contacted with oxygen, air, or other oxidizing media.

EXAMPLE The invention is further demonstrated by the following illustrative example.

A series of compounds was assayed for effectiveness against bacteria, yeasts, and molds, using the following test procedure.

All compounds were initially tested at a concentration of 200 or 500 p.p.m. In some cases, assays also were conducted with varying amounts of the compounds to determine the minimum concentration thereof required to inhibit growth.

Plates were prepared by adding a measured amount of the candidate compound in acetone solution to ml. of sterilized medium, mixing thoroughly, pouring into 60 x mm. plastic petri dishes, and allowing the gel to set and TABLE IIL-MINIMAL INHIBITORY CONCENTRATIONS (IN age overnight. The plates were then inoculated with the test organisms. In the case of bacteria and yeasts, the inoculation was done by the Lederberg and Lederberg (J. BacterioL, 63, 399, 1952) replica plating technique, applying nine bacteria or seven yeasts on each plate. In the case of molds, drops of homogenized culture were placed on the surface of the plates, applying three or four molds per plate. The media used were: plate count agar (Difco), ph 7.0, for bacteria; and potato dextrose agar (Difco), pH 5.6, for yeasts and molds. Inoculated control plates were also prepared with media containing the largest amount of acetone (0.8 ml.) used in the preparation of the test plates. We have determined that acetone has a minimal influence on the growth of organisms in this procedure. The plates were incubated at 28 for 1-5 days and evaluated by comparison with the controls.

The results obtained are summarized in the following tables. In these tables the compounds are designated in abbreviated manner using DH for dihydro, CA for cinnamyl, and PH for phenol. Thus, for example, dihydro-4-cinnamyl-phenol is designated as DH-4-CA-PH; dihydro-2-cinnamyl-4-isopropylphenol is designated as DH-2-CA-4-isopropyl-PH.

TABLE L-M'INIMAL INHIBITORY CONCENTRATIONS N P.P.M. OF DIHYDROCINNAMYL PHENOLS ON GROWTH OF GRAM-POSITIVE BACTERIA Bacillus Staph.

cereus Sarcina aureaa Strep. Compound 06 lutea SG8A Zactts DH-4-GA-PH 25 25-50 25-50 25 12 25 25 12 12 12 12 12 12 12 12 12 12 12 12 12 25 12 12 12 12 25 12-25 12 PH DH-2-CA-4-methoxy-PH 13 12 25-50 25 The symbol indicates complete inhibition of growth at a concentration of 500 p.p.m. The compound in question was not tested at lower concentration.

TABLE II Minimal inhibitory concentration (in p.p.m.) of dihydro- 4 cinnamyl phenol on growth of gram-negative bacteria i Minimal inhibitory conc.

Bacteria: of DH-4-CA-PH, p.p.m. Alcaligenes faecalis Bl 200 Escherichia coli ML30 200 Pseudomonas aeruginosa III 200 Salmonella typhimurium Tm 1 200 Serratia marcescens 200 .P.M.) OF VARIOUS DIHY- P DROCINNAMYL PHENOLS ON GROWTH OF YEASIS Saccharo- Zygosacchw Pzchiamycea Torula romyces Candida chodati var. cerevistae var. uttlt's japonicua tropicalls fermentans Hamenula ellipsoz'deus NRRL Compound C-124 C-147 (3-238 anomala Y- te Y-66 DH-t-CA-PH 25 25 50 50 25 25 DH-2-C -PH 12 12 5O 25 12 25 DH-2-CA-4-methyl-PH.--.- 12 12 25 12 DH-4-CA-2-methy1-PH..--. 12 12 200 12 200 DH-2-OA-4ethy1-PH 12-25 12-25 50 100 DH-2-OA-4-methoxy-PH- 25 25 50 25 200 The symbol indicates no growth inhibition at cone. of 500 p.p.m. The term 200" indicates growth not inhibited with 200 p.p.m. (hlghest concentration tested in this case).

TABLE IV.--M1NIMAL CONCENTRATIOgg Aspergz'llus Aspergz'llus Penicillium Rhizopua Botrytis Bysaochlamys {lama m'gcr chryeogenum semi cinerm fulva Alternaria Compound NRR 8145 A-7705 52 N RRL 2868 NRRL 3492 NRRL 3493 sp.

DH-4-CA-PH 200 50-100 25 50 12-25 50 25 50 50 25 25 12 25 25 200 200 200 100 200 200 100 200 200 25 50 25 25-50 2550 200 100-200 200 200 DH-2CA4-isopt0pyl-PH 200 200 200 100 DH2-CA4-methoxy-PH 200 100 200 200 200 The symbol indicate no growth inhibition at concentration of 500 p.p.m. The term 200 indicates growtu not inhibited at 200 p.p.m. (highest contenstration tested in this case).

Having thus described the invention, what is claimed is:

1. A process for inhibiting the growth of microorganisms selected from the group consisting of bacteria, yeasts, and molds in a substance normally subject to spoilage by said microorganisms, which comprises applying to said substance an effective microbial-growthinhibiting amount of a compound of the structure- R (CR2) 7. The process of claim 1 wherein the compound is dihydro-Z-cinnamyl-4-n-propylphenol.

8. The process of claim 1 wherein the compound is dihydro-2-cinnamyl-4-isopropylphenol.

9. The process of claim 1 wherein the compound is dihydro-4-cinnamyl-3-methyl-6-isopropylphenol.

References Cited UNITED STATES PATENTS 8/1970 Reinert et al. 424-346 OTHER REFERENCES Hurd et al., Jour. Am. Chem. Soc., vol. 59, pp. 107- 109.

Barnes et al., Tetrahedron, vol. 21, pp. 2707-2715.

Jurd, Experientia, vol. 24, pp. 858860.

Jurd, Tetrahedron, vol. 25, pp. 1407-1416.

Jurd, Tetrahedron, Letters N0. 33, pp. 2863-2866.

Thakar et al., Chem. Abst., vol. 63 (1965), pp. 571- 572.

JEROME D. GOLDBERG, Primary Examiner A. J. ROBINSON, Assistant Examiner US. Cl. X.R. 252-404; 424-341 

